Isolation of microvesicles and exosomes by microfiltration and estimation of normal reference range in blood plasma.

Current protocols for the isolation of microvesicles (MVs) and exosomes, which in the main focus on differential centrifugation, vary considerably (1). In an attempt to set a new standard, we describe a filtration protocol for isolating phosphatidylserine-positive MVs (larger than 100 nm in diameter) and exosomes. The key preparative step to successfully isolate both MVs and exosomes to a high degree of purity was a gentle sonication to break up exosome clumps. Filtration through a 100 nm pore size Millipore filter allowed for collection of exosomes in the filtrate. The larger MVs could then be recovered from the filter. Annexin V-PE MVs were sized and quantified using Polysciences Polybead Microspheres (200 nm) and BDTrucount tubes, respectively on a FACS CaliburTM flow cytometer. The normal reference range from normal human donors was found to be 0.51-2.82 x105 MVs/ml. Freeze/thawing of samples had little effect on MV counts and with age MV levels seemed only marginally reduced. Fasting status also affected MV levels, appearing up to 3-fold higher in fasting individuals. Smokers had lower MV counts and nicotine reduced MV release from THP-1 cells.